ER membranes exhibit phase behavior at sites of organelle contact

The endoplasmic reticulum (ER) is the site of synthesis of secretory and membrane proteins and contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner. How the ER spatially segregates its numerous and diverse functions, including positioning nanoscopic contact sites with other organelles, is unclear. We demonstrate that hypotonic swelling of cells converts the ER and other membrane-bound organelles into micrometer-scale large intracellular vesicles (LICVs) that retain luminal protein content and maintain contact sites with each other through localized organelle tethers. Upon cooling, ER-derived LICVs phase-partition into microscopic domains having different lipid-ordering characteristics, which is reversible upon warming. Ordered ER lipid domains mark contact sites with ER and mitochondria, lipid droplets, endosomes, or plasma membrane, whereas disordered ER lipid domains mark contact sites with lysosomes or peroxisomes. Tethering proteins concentrate at ER–organelle contact sites, allowing time-dependent behavior of lipids and proteins to be studied at these sites. These findings demonstrate that LICVs provide a useful model system for studying the phase behavior and interactive properties of organelles in intact cells.

Insulin-like Growth Factor-1 Regulates the Mechanosensitivity of Chondrocytes by Modulating TRPV4

ABSTRACTBoth mechanical and IGF-1 stimulation are required for normal articular cartilage development and maintenance of the extracellular matrix. While much effort has been made to define the signaling pathways associated with these anabolic stimuli, we focused on how these pathways interact to regulate chondrocyte function. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. However, the mechanism by which TRPV4 is mechanically gated or regulated is not clear. In this study we propose that insulin-like growth factor 1 (IGF-1), which is important in regulating matrix production during mechanical load, modulates TRPV4 channel activity. Our studies indicate that IGF-1 reduces hypotonic-induced TRPV4 currents, and intracellular calcium flux by increasing stress fiber formation and apparent cell stiffness. Disruption of F-actin following IFG-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Furthermore, we highlight that IGF-1 suppresses TRPV4 mediated calcium flux through the MAP7 binding domain (aa. 798-809), where actin binds to the TRPV4 channel. IGF-1 treatment differentially influences the intracellular calcium flux of HEK 293 cells stably expressing either wild-type or mutant (P799L or G800D) TRPV4 during hypotonic challenge. A key down-stream response to mechanical stimulation of chondrocytes is ATP release. Data here indicate that activation of TRPV4 through hypotonic swelling induces ATP release, but this release is greatly reduced with IGF-1 treatment. Taken together this study indicates that IGF-1 modulates TRPV4 channel response to mechanical stimulation by increasing cell stiffness. As chondrocyte response to mechanical stimulation is greatly altered during OA progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

New Tracer for Magnetic Particle Imaging - SPIONs encapsulated in RBCs

Superparamagnetic iron oxide nanoparticles, so called SPIONs, are used as tracers in medical imaging, e. g. for magnetic particle imaging (MPI) or magnetic resonance imaging (MRI). Since the half-life time of the SPIONs in the bloodstream is quite short because they are quickly absorbed by the reticuloendothelial system (RES), the particles are introduced into human red blood cells (RBCs) to increase their half-life time in the blood circulation. The hypotonic swelling procedure is used to incorporate the particles into the RBCs. Before the SPIONs are introduced into the RBCs, they are fluorescent labelled. To evaluate the result transmission electron microscopy, magnetic particle spectroscopy and fluorescence microscopy are used. Fluorescein isothiocyanate and rose Bengal were chosen as fluorescent dyes because their biocompatibility is guaranteed. The results suggest that the method hypotonic swelling can be used to successfully introduce the nanoparticles into RBCs and that the magnetic properties of the particles which are necessary for imaging are not influenced.

Differential volume regulation and calcium signaling in two ciliary body cell types is subserved by TRPV4 channels

Fluid secretion by the ciliary body plays a critical and irreplaceable function in vertebrate vision by providing nutritive support to the cornea and lens, and by maintaining intraocular pressure. Here, we identify TRPV4 (transient receptor potential vanilloid isoform 4) channels as key osmosensors in nonpigmented epithelial (NPE) cells of the mouse ciliary body. Hypotonic swelling and the selective agonist GSK1016790A (EC50 ∼33 nM) induced sustained transmembrane cation currents and cytosolic [Ca2+]i elevations in dissociated and intact NPE cells. Swelling had no effect on [Ca2+]i levels in pigment epithelial (PE) cells, whereas depolarization evoked [Ca2+]i elevations in both NPE and PE cells. Swelling-evoked [Ca2+]i signals were inhibited by the TRPV4 antagonist HC067047 (IC50 ∼0.9 μM) and were absent in Trpv4−/− NPE. In NPE, but not PE, swelling-induced [Ca2+]i signals required phospholipase A2 activation. TRPV4 localization to NPE was confirmed with immunolocalization and excitation mapping approaches, whereas in vivo MRI analysis confirmed TRPV4-mediated signals in the intact mouse ciliary body. Trpv2 and Trpv4 were the most abundant vanilloid transcripts in CB. Overall, our results support a model whereby TRPV4 differentially regulates cell volume, lipid, and calcium signals in NPE and PE cell types and therefore represents a potential target for antiglaucoma medications.

Vascular endothelial cell membranes differentiate between stretch and shear stress through transitions in their lipid phases

Vascular endothelial cells (ECs) respond to the hemodynamic forces stretch and shear stress by altering their morphology, functions, and gene expression. However, how they sense and differentiate between these two forces has remained unknown. Here we report that the plasma membrane itself differentiates between stretch and shear stress by undergoing transitions in its lipid phases. Uniaxial stretching and hypotonic swelling increased the lipid order of human pulmonary artery EC plasma membranes, thereby causing a transition from the liquid-disordered phase to the liquid-ordered phase in some areas, along with a decrease in membrane fluidity. In contrast, shear stress decreased the membrane lipid order and increased membrane fluidity. A similar increase in lipid order occurred when the artificial lipid bilayer membranes of giant unilamellar vesicles were stretched by hypotonic swelling, indicating that this is a physical phenomenon. The cholesterol content of EC plasma membranes significantly increased in response to stretch but clearly decreased in response to shear stress. Blocking these changes in the membrane lipid order by depleting membrane cholesterol with methyl-β-cyclodextrin or by adding cholesterol resulted in a marked inhibition of the EC response specific to stretch and shear stress, i.e., phosphorylation of PDGF receptors and phosphorylation of VEGF receptors, respectively. These findings indicate that EC plasma membranes differently respond to stretch and shear stress by changing their lipid order, fluidity, and cholesterol content in opposite directions and that these changes in membrane physical properties are involved in the mechanotransduction that activates membrane receptors specific to each force.

Abstract 99: Hypotonic Swelling Promotes Nitric Oxide Release in Cardiac Ventricular Myocytes

Cardiac myocyte swelling occurs in multiple pathological situations and in particular contributes to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction. We investigated whether hypotonic swelling promotes nitric oxide (NO) release in cardiac myocytes and if so, whether it impacts on swelling induced contractile dysfunction. Perfusing rat cardiac myocytes, loaded with the NO sensor DAF-FM, with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient amplitude and significantly increased DAF-FM fluorescence. When cells were exposed to the HS supplemented with 2.5 mM of the NO synthase inhibitor L-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the NOS1 inhibitor, Nitroguanidine. In addition, Colchicine (an inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either L-NAME, Nitroguandine or the guanylate cyclase inhibitor, ODQ, suggesting that NOS1-derived NO provides contractile support via a GMP-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and the HS-induced increment in ryanodine receptor (RyR2) phosphorylation at site Ser2808 suggesting that in the context of hypotonic swelling, cGMP may contribute to preserve contractile function by enhancing SR Ca2+ release. Our findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may be cardioprotective by reducing the extent of contractile dysfunction associated with hypotonic swelling.